Defining how neurons are assembled into functional circuits will provide insights into diverse disorders of the nervous system and may suggest strategies to promote neuronal repair. Slit and its Robo receptors comprise an evolutionary conserved family of signaling molecules that play critical roles in regulating neuronal development; however, the understanding of how Robo receptors are regulated and how they signal to direct axon guidance is incomplete. These are important questions because perturbations of Robo signaling are implicated in diseases of nervous system development. Slits and Robos also play essential developmental roles in non- neuronal tissues and Robo mis-regulation is associated with several kinds of cancer. This proposal will define the molecular mechanisms underlying Robo regulation and signaling using the genetically tractable Drosophila embryonic nervous system as a model. Using state of the art molecular, genetic, biochemical and cell biological approaches we will continue our ongoing investigation of Robo receptor biology. Specifically, we propose to characterize the role of Robo receptor cleavage and endocytosis during midline axon repulsion. Genetic evidence indicates that endocytosis positively regulates repulsion, while receptor proteolysis appears to negatively regulate repulsion. We will explore how these processing and trafficking events are spatially and temporally coordinated to control Robo receptor signaling. Additionally, we will investigate a novel role for the Robo2 receptor in antagonizing Slit-Robo signaling to allow midline crossing. Biochemical and genetic evidence supports the model that Robo2 binds to Robo to inhibit repulsive signaling and we will define the underlying mechanism using a combination of genetic, biochemical and biophysical techniques. Finally, we will investigate how Robo2 and Robo3 regulate the medial-lateral position of axons within the CNS by 1) exploring the biochemical and cell biological mechanisms through which Robo2 and 3 direct longitudinal pathway choice and 2) identifying new factors that work with Robo2 and 3 to mediate this guidance activity. Preliminary evidence indicates that distinct biochemical properties imparted by Robo receptor immunoglobulin domains confer specific guidance outputs. We will mutate these domains and determine how these manipulations affect the in vivo functions of the receptors. Our proposed research will 1) define new concepts in the molecular biology of axon guidance, 2) inform studies of homologous proteins in mammalian systems and 3) provide pharmacologic and genetic strategies to manipulate receptor signaling.